Body temperature drops dramatically during hibernation, but the heart retains the ability to contract and is resistant to induction of arrhythmia. Although adaptive changes in the heart prior to hibernation may be involved in the cold-resistant property, it remains unclear whether these changes are sufficient for maintaining cardiac pulsatility under an extreme hypothermic condition. We forcibly induced hypothermia in Syrian hamsters by pentobarbital anesthesia combined with cooling of the animals. This allows reproduction of a hypothermic condition in the absence of possible hibernation-specific reactions. Unlike hypothermia in natural hibernation, the forced induction of hypothermia caused atrioventricular block. Furthermore, J-waves, which are typically observed during hypothermia in nonhibernators, were recorded on an ECG. The origin of the J-wave seemed to be related to irreversible injury of the myocardium, because J-waves remained after recovery of body temperature. An abnormal ECG was also found when hypothermia was induced in hamsters that were well adapted to a cold and darkened environment or hamsters that had already experienced hibernation. These results suggest that acclimatization prior to hibernation does not have a crucial effect at least on acquisition of cardiac resistance to low temperature. In contrast, an abnormal ECG was not observed in the case of hypothermia induced by central administration of an adenosine A1-receptor agonist and subsequent cooling, confirming the importance of the adenosine system for inducing hibernation. Our results suggest that some specific mechanisms, which may be driven by a central adenosine system, operate for maintaining the proper cardiac pulsatility under extreme hypothermia.
some rodents undergo hibernation to survive a severe environment during winter. During hibernation they regulate their body temperature down to only a few degrees above ambient temperature to save in energy expenditure (6, 10). Circulation and respiration are also well regulated in hibernating animals but are suppressed at much lower rates than these in euthermic counterparts (14, 23, 26). Furthermore, mammalian hibernators exhibit remarkable cardiac resistance to low body temperature (17), whereas nonhibernating mammals develop ventricular dysfunction and arrhythmias, such as atrioventricular block and ventricular fibrillation (22, 34).
Cardiac myocytes of hibernating mammals are characterized by a reduced activity of L-type Ca2+ channels in the cell membrane (1, 7, 38) and a concomitant enhancement of Ca2+ uptake by the sarcoplasmic reticulum (2, 20, 37). These adaptive changes would help in preventing excessive Ca2+ entry and its overload and in keeping the resting levels of intracellular Ca2+ stable (37). In accordance with this, expression of Ca2+-handling proteins, such as L-type Ca2+ channel α1C-subunit, sarco(endo)plasmic reticulum Ca2+-ATPase 2a and phospholamban, has been shown to change in hibernating animals in a manner consistent with functional changes (3, 38). Moreover, expression of functional proteins related to contractility (e.g., myosin heavy chain-α, ventricular myosin light chain, troponin C) and proteins involved in synchronous contraction (e.g., connexin43) can be upregulated or downregulated appropriately in hibernating animals (3, 9, 31). Importantly, the onset of these changes precedes the onset of hibernation (18, 31), indicating that these changes in gene expression and subsequent functional remodeling are preparatory processes for entering hibernation and are therefore indispensable for acquiring cold resistance.
However, it remains unclear whether these adaptive changes prior to hibernation are sufficient for maintaining cardiac pulsatility under an extreme hypothermic condition during hibernation. To address this question, we have focused on hypothermia induced by pentobarbital sodium (12, 16, 33). By combining pentobarbital sodium anesthesia with cooling of the animal, forced hypothermia that is comparable to that in hibernating animals can be successfully produced. This procedure may allow us to reproduce a hypothermic condition in vivo without promoting possible neuroautonomic functions that would usually be triggered in natural hibernation. If the adaptive changes exclusively contribute to cold tolerance of the heart, heart pulsatility of well-adapted hamsters can be maintained appropriately not only during natural hibernation but also during a forcibly induced hypothermic condition. Therefore, artificial hypothermia was induced in hamsters before and after exposure to an environment that is appropriate for induction of adaptive changes, and their ECGs were compared with those of hamsters in natural hibernation. In addition, central administration of an adenosine A1-receptor agonist was employed to induce hypothermia, because the receptor-mediated signals have been demonstrated to act as a trigger of hibernation in hamsters (35). Our results indicate that some specific mechanisms, which are independent of adaptive changes prior to hibernation, operate for maintaining proper cardiac pulsatility under a condition of extreme hypothermia in hamsters.
MATERIALS AND METHODS
Animals: breeding and induction of hibernation.
Male and female Syrian hamsters obtained by mating of hamsters that had previously undergone hibernation were used in this study. Animals were maintained at an ambient temperature of 24–25°C with a 12:12-h light-dark cycle (lights on 0700–1900) and given free access to food and water until 8 wk of age. Then, they were transferred to a constantly darkened cold (2°C) room and acclimated to this condition for 2 mo. During this acclimation period, 4 animals were housed together per cage. No hamster entered hibernation when multiple animals were kept in a cage. To induce hibernation, each acclimated hamster was housed in an individual cage. This procedure allowed the hamster to go into hibernation within a few weeks with a relatively high probability (over 80%) compared with our previous method (30). Care and experimental procedures were approved by the Animal Care and Use Committee of Gifu University.
Intracerebroventricular administration of N6-cyclohexyladenosine.
The hamsters were anesthetized with pentobarbital sodium (60 mg/kg ip) and fixed in a stereotaxic frame (SR-6N; Narishige Scientific Laboratory, Tokyo, Japan). The scull of each hamster was exposed, and the bregma was located, and then a stainless-steel guide cannula (AG-6; Eicom, Kyoto, Japan) was unilaterally implanted into the lateral ventricle (−0.6 mm AP, 2.6 mm L, 4.0 mm DV), according to the hamster brain atlas (27). The guide cannula was fixed to the calvaria with miniature stainless screws and acrylic dental cement and was plugged with a solid dummy cannula (AD-6; Eicom). Hamsters were allowed at least 1 wk for recovery. N6-cyclohexyladenosine (CHA) (Sigma, St. Louis, MO) was dissolved in an artificial cerebral fluid (aCSF; in mM): 125 NaCl, 2.5 KCl, 1.18 MgCl2·6H2O, 2.0 Na2HPO4, adjusted to pH 7.4. One nanomole of CHA in 5 μl of aCSF was administered through a microinjection cannula (AMI-6; Eicom).
Induction of hypothermia.
To induce extreme hypothermia, the hamsters were placed in an ambient temperature of 2°C after administration of CHA (1 nmol icv) or pentobarbital sodium (80 mg/kg ip). By this procedure, body temperature of the animals was lowered to less than 10°C within 3 h. The dose of CHA used in this study has been expected to be specific for the adenosine A1-receptor (21). A higher dose of pentobarbital sodium (80 mg/kg) than that used for a surgical operation (i.e., 60 mg/kg) was needed for inducing extreme hypothermia. In the preliminary experiment, almost all hamsters were successfully aroused after the high-dose pentobarbital administration under a euthermic condition. In some experiments, hypothermia was induced in hamsters that had been acclimated to a cold and darkened condition. We selected the animals from a colony that was under the process of inducing hibernation. Since the timing of selection of the acclimated hamsters was after induction of hibernation in some animals of the same colony, the nonhibernated hamsters were assumed to have completed almost all of the necessary adaptation for hibernation. In other words, these hamsters were expected to enter hibernation within a week.
To examine whether extreme hypothermia can also be induced in a nonhibernator or not, male Wistar rats with or without cold acclimation were used. The protocol giving cold acclimation for rats was same as that used in hamsters. Rats were kept under a constantly darkened cold (2°C) room and acclimated to this condition for 2 mo, and then each rat was housed in an individual cage prior to experiments. The procedure for the induction of hypothermia was the same as that for hamsters, but a lower dose of pentobarbital (50 mg/kg ip) was given to rats because the dose suitable for hamsters (80 mg/kg) is lethal for rats.
Measurement of body temperature.
Body temperature was monitored using a telemetry system (DAS-5002; BioMedic Data Systems, Seaford, DE). The transmitter (IPTT-200; BioMedic Data Systems) used for measuring body temperature was implanted subcutaneously between scapulas. This system enabled measurement of body temperature without giving any noise on ECG recording. The transmitter is certified in its use above 30°C, but in our preliminary experiment the body temperature measured by this transmitter was comparable to that by a rectal thermometer even below 30°C.
After induction of hibernation or hypothermia, an ECG was recorded from needle electrodes placed across the chest with a ground electrode placed at one hindlimb. In contrast to the standard limb lead, the method employed in the present study is inadequate for analyzing amplitudes of ECG waves, but the rhythmicity of the heartbeats can be properly analyzed. The signal was amplified and displayed on a recorder (M1117A; Hewlett Packard, Palo Alto, CA). After recordings of ECG during the hypothermic conditions, all animals were transferred to a warm room kept at 24°C and warmed by using a heating blanket (Homeothermic blanket system, Harvard apparatus, Holliston, MA) to allow recovery of body temperature.
All values are expressed as means ± SD. Statistical differences between the groups were determined by ANOVA followed by Fisher's test. P < 0.01 was considered to be significant.
ECG of hibernating hamsters in summer and winter.
Hibernation, as judged by disappearance of locomotor activity, lowered body temperature and reduced respiratory rate for more than 24 h, was successfully induced both in summer and winter. The average body temperatures of hibernating hamsters in summer and winter were comparable (5.0 ± 0.9°C and 5.5 ± 0.3°C, respectively). The ECG tracings recorded from these animals were similar (Fig. 1). Periodic cycles of heartbeats were recorded on ECG in hibernating hamsters both in summer and winter, although the heart rates were much lower than those in nonhibernating animals (Table 1). A detailed analysis of ECG recordings showed that P-Q intervals and duration of QRS complex in hibernating hamsters were extended compared with those in nonhibernating animals (Table 1). There was no seasonal difference in these parameters. Abnormal ECGs related to conduction block (e.g., P-wave lacking corresponding QRS complex), arrhythmia, or other forms of ectopy were not observed in either season.
ECG during the artificial hypothermia induced by pentobarbital and cooling.
We then addressed the question of whether maintenance of cardiac pulsatility under an extreme hypothermic condition depends on the natural ability of hamster hearts or whether it is achieved by some specific mechanisms that operate during hibernation. To reduce body temperature forcibly, hamsters without acclimatization to a cold and darkened environment were anesthetized with pentobarbital sodium (80 mg/kg ip) and cooled in a refrigerator (2°C). This procedure reduced body temperature to less than 10°C, a level comparable to that in hibernating animals, within 3 h. The values of ECG parameters during this hypothermia show no significant differences to those during hibernation (Table 1). However, atrioventricular block occurred in 90% of the hypothermic animals (Fig. 2), which hindered analysis of P-Q interval in this group of animals. Furthermore, an abnormal ECG wave called J-wave or Osborne wave was clearly observed in all recordings during hypothermia induced by pentobarbital administration and cooling (Fig. 2).
We then applied the same procedure for induction of artificial hypothermia in rats. As body temperature decreased, heart rates of the rats consistently dropped. However, in contrast to hamsters, rats showed cardiac arrest when their body temperature fell below 20°C (data not shown). Acclimatization to a cold and darkened environment had no effect on the hypothermia-induced cardiac arrest in rats.
ECG during artificial hypothermia in acclimatized hamsters.
To determine whether the adaptive changes are sufficient for the heart to acquire cold-resistant ability, artificial hypothermia was induced in hamsters acclimated to a cold and darkened environment. The hamsters used in this experiment were selected from a colony in which some animals had begun to hibernate and therefore assumed to have completed adaptive changes. Nevertheless, signs of atrioventricular block and J-wave were recorded on their ECGs (Fig. 3A).
We then induced artificial hypothermia in hamsters that had already experienced hibernation. Since hamsters under successful hibernation were forcibly aroused by gentle touching and warming and used within a few hours, it can be assumed that the animals had completed all of the required adaptations. Even in those hamsters, ECG recordings showed the presence of atrioventricular block. J-waves were also observed as in the case of unacclimated animals (Fig. 3B).
ECG after injection of pentobarbital to hibernating hamsters.
To examine the possibility that the abnormal ECG observed in artificial hypothermia (Fig. 2) is due to unexpected effects of pentobarbital, we injected hibernating hamsters with anesthetic. As shown in Fig. 4, injection of pentobarbital to the hibernating hamster did not cause atrioventricular block or J-wave generation. The ECG was recorded about 3 h after pentobarbital injection to fit the time-course with the recording time point in the artificial hypothermia.
ECG during hypothermia induced by CHA.
It has been demonstrated that activation of the adenosine A1-receptor is important for the generation of hypothermia in the entrance phase of hibernation (35). We, therefore, tried to produce a hibernation-like hypothermia by intracerebroventricular injection of an adenosine A1-receptor agonist, CHA. Administration of CHA into the lateral ventricle followed by cooling the hamsters in a refrigerator (2°C) resulted in extreme hypothermia. P-waves appeared periodically, and each of them was accompanied by QRS complex in a constant position (Fig. 5). The values of ECG parameters were not significantly different from those of hibernating hamsters (Table 1). Atrioventricular block in CHA-induced hypothermia was observed only in one of the six animals despite the fact that the hamsters injected with CHA had not undergone acclimatization for cold and darkness.
ECG after recovery from hibernation or artificial hypothermia induced by pentobarbital and cooling.
To determine whether abnormal ECG observed in artificial hypothermia induced by pentobarbital anesthesia and subsequent cooling is reversible, the hamsters were allowed to recover. In contrast to natural hibernation, from which all hamsters were successfully aroused, 8 of the 10 unacclimatized and 7 of the 10 acclimatized animals failed to recover from the artificial hypothermia, even though they were warmed up. The mean time until cardiac arrest from pentobarbital administration was 326 ± 40 min. In some cases in which arousal succeeded, J-wave, which developed during extreme hypothermia, was still observed on the ECG (Fig. 6B). This was in contrast to hamsters that recovered from hibernation, in which no abnormal sign was recorded on ECG (Fig. 6A).
It is well known that seasonal hibernators, e.g., Spermophilus richardsonii and Tamias sibiricus asiaticus, rarely hibernate in summer, even if they are placed in a cold (4°C) condition (19). This indicates that the endogenous circannual rhythm plays a critical role in the induction of hibernation in seasonal hibernators. In contrast, hamsters hibernated even in summer when they were placed in a condition suitable for induction of hibernation. We failed to find any differences between the hibernation in summer and that in winter, at least in the parameters examined in this study (Table 1). Therefore, the endogenous circannual rhythm might only have a minor contribution, if any, to the induction of hibernation in this species. It should be noted, however, that the present study does not necessarily rule out the possibility of involvement of the circannual rhythm in the induction of hibernation in hamsters. It may be appropriate to consider that the relative importance of endogenous and environmental factors varies among species and that this variation is a determinant for seasonal or nonseasonal hibernators.
Cardiac contractility was maintained in the hamsters in which extreme hypothermia has been forcibly induced by pentobarbital and cooling (Fig. 2). This is in sharp contrast to nonhibernators, in which cardiac arrest is usually induced at a low temperature (5, 8, 15). In fact, our procedure for inducing artificial hypothermia in hamsters was lethal in rats. Therefore, an ability to maintain cardiac contractility under an extreme hypothermic condition can be recognized as an inherent feature of hibernators. However, appropriate cardiac pulsatility under extreme hypothermia may not totally depend on the inherent ability of hibernators as discussed below.
It has been reported that atrial arrhythmias are occasionally observed in the entrance phase of natural hibernation (26). Heart rate is significantly greater during eupnea than during apnea when animals are breathing episodically, and these atrial arrhythmias are eliminated by bilateral vagotomy (14, 25, 26), indicating that the arrhythmias in entrance phase of hibernation are associated with an activation of autonomic nerves. In the present study, we tried to detect damage of the myocardium and/or conducting system by using ECG. For this purpose, it is preferable to use experimental conditions in which autonomic influences are minimized. We, therefore, chose the deeply hibernating stage rather than the initial stage of hibernation.
We found P-waves not followed by QRS complex, which is a sign of atrioventricular block, in the hamsters in which hypothermia was forcibly induced (Fig. 2, A and B). In addition, an abnormal ECG wave, J-wave, was obvious in the forced hypothermia (Fig. 2). J-wave is a wave located at the point of the end of the QRS complex and occupies the initial part of the ST segment (13). This ectopic wave is typically described in hypothermia in nonhibernating mammals (4, 29). The origin of the J-wave during hypothermia has been attributed to injury current, delayed ventricular depolarization and early repolarization, tissue anoxia, and acidosis (4). Possible harmful effects of pentobarbital under the extreme hypothermic condition, if any, may not be a major cause for the appearance of the J-wave, as well as conduction block, since injection of pentobarbital to the hibernating hamsters had no effect on the ECG (Fig. 4). This is consistent with a reported observation by Milsom et al. (25), who demonstrated that pentobarbital injection to deeply hibernating squirrels failed to elicit abnormal ECGs such as J wave and/or atrioventricular block. Therefore, the fact that the J-wave, as well as abnormal ECG signs related to conduction block, was not observed in natural hibernation (present study and Ref. 23) can be rationally explained by the operation of regulatory mechanisms during natural hibernation to coordinate the cardiac conducting system properly and to prevent cardiac impairment caused by hypothermia.
There was no sign of hypoxia in gross observation of the color of skin and mucosa in all hypothermic conditions used in the present study, suggesting the absence of severe hypoxia. However, we cannot totally exclude possible involvement of hypoxia, in addition to hypothermia, in the incidence of the abnormal ECG in the forced hypothermia. Further study is needed to clarify the factors that cause ECG abnormalities.
A comprehensive analysis of mRNAs before and during hibernation in the thirteen-lined ground squirrel revealed that about 50 genes are differentially regulated in the heart (3). Several of the differentially expressed genes encode proteins that likely play a role in altering contractility and Ca2+ handling in the heart of the hibernating animal. Furthermore, gap junctional protein connexin 43, which is associated with potential propagation, is upregulated in the heart of hibernating hamsters (31). These differential gene expressions may provide an explanation for the cold-resistant property of the hibernators. It can, therefore, be speculated that lack of some changes in molecular expression or inadequate regulation of these molecules is involved in the occurrence of abnormal ECGs during the forcibly induced extreme hypothermia. It should be noted, however, that gene expressions during the acclimatization period prior to hibernation do not have a crucial effect at least on acquisition of cardiac resistance to low body temperature, because hamsters that were expected to be ready for entering hibernation, as well as hamsters that had already experienced hibernation, showed abnormal ECGs when extreme hypothermia was forcibly induced by pentobarbital and cooling (Fig. 3). Hibernators probably possess mechanisms for maintenance of coordinated cardiac function during hibernation in nature, and the mechanisms would be triggered during the initiation of hibernation. Recently, it has been demonstrated that local autocrine and paracrine factors are released within the heart in response to ischemia and protect the heart during ischemia-reperfusion injury (24). The release of such protective factors within the heart would be a candidate for the mechanism that operates at an early stage of hibernation.
It has been demonstrated that adenosine A1-receptor-mediated signals play a role in the induction of hibernation (35). We, therefore, thought it would be interesting to determine whether stimulation of the adenosine A1-receptor-mediated pathway in the brain before inducing hypothermia prevents the abnormality in the ECG. Cooling of the hamster after central administration of the receptor agonist CHA reduced body temperature, confirming the involvement of the adenosine A1-receptor-mediated pathway in the induction of hibernation. Under this hypothermic condition, J-wave was not observed (Fig. 5). The dose of CHA used in the present study is expected to be specific for the adenosine A1-receptor in rodents (21). Therefore, it can be assumed that the signals mediated by the adenosine A1-receptor trigger the mechanisms for maintenance of coordinated cardiac pulsatility during hibernation. It should be noted, however, that the present study does not necessarily rule out the possible involvement of other types of receptors. The involvement of receptors such as adenosine A2-receptor remains to be examined. It has been reported that adenosine in the nucleus of the solitary tract, a primary integrative center for cardiovascular reflex, plays a role in modulating arterial pressure, heart rate and vascular conductance by tuning activity of the sympathetic and parasympathetic systems. (32, 36) These comprehensive effects of adenosine on cardiovascular function may be one of the mechanisms of the cardioprotective effects during hibernation. Further studies are needed to elucidate the precise mechanisms of the adenosine-mediated cardiac-protective actions. It is noteworthy that the hamster in which hypothermia was induced by CHA was not exposed to a cold and darkened environment at all. This provides further evidence supporting the notion that acclimatization prior to hibernation is not necessary to maintain cardiac pulsatility at least in an early stage of hypothermia.
Most of the hamsters in which hypothermia was induced by anesthetics and cooling did not survive even though their body temperature had recovered by warming. Arousal was observed in rare cases, but the J-wave was still present in their ECG. These results indicate that extreme hypothermia irreversibly injures the heart in hibernators as in nonhibernators. Considering the fact that there was no abnormal ECG after recovery from natural hibernation, it is likely that some defense mechanisms operate in the entire process of hibernation. The precise mechanisms responsible for protection of the heart remain to be clarified.
Perspectives and Significance
The present results indicate that cold tolerance of the heart in the hibernating hamster is not totally dependent on the adaptive changes that occur during acclimatization but is supported by mechanisms that operate during the entry to hibernation. Although the mechanisms are largely unknown at present, adenosine A1-receptor-mediated signals in the brain are thought to be involved. It has been reported that stimulation of adenosine A1-receptor in the heart enhances the expression of genes involved in cardiac calcium homeostasis (11) and improves the cardiac function after ischemia/reperfusion injury (28). In addition to the beneficial actions of peripheral adenosine A1-receptor stimulation, our results suggest that central adenosine A1-receptor has protective effects on heart function. If the underlying mechanisms of the central action of adenosine are not specific to hibernators but are also employed by nonhibernators, including humans, centrally penetrant adenosine A1-agonists may be candidates for new drugs, especially for reperfusion injury in ischemic diseases.
This work was supported by a Grant-in-Aid for Scientific Research (The 21st Century Center-of Excellence Program) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (E-1).
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